CRISPR-Mediated Knockout of CD55 in CD34+ Cells

Two sgRNAs targeting human CD55 exons were designed using the Broad Institute’s GPP sgRNA design portal and synthesized as chemically modified sgRNAs by Synthego. CD55-Cr1 is predicted to recognize a sequence in exon 1 of CD55 and has the sequence: GGGCCCCUACUCACCCCACA. CD55-Cr8 is predicted to recognize a sequence in exon two and has the sequence: CUGGGCAUUAGGUACAUCUG. Experiments using dual sgRNAs typically produce large deletions between the Cas9 binding sites as well as smaller indels, leading to frameshift mutations (Mandal et al., 2014 (link)). Ribonucleoprotein (RNP) complexes containing one or both sgRNAs were prepared by slowly adding 300 pmol of each sgRNA to 150 pmol Cas9 protein in a 10 µl final volume with nuclease-free water and incubating at room temperature for 10 min. On day 2 after thawing CD34 + cells, the RNP complexes were added to 1 × 10⁵ cells in 40 µl of P3 nucleofection buffer from the 4D-Nucleofector X kit (Lonza). Half of the mixture was loaded to each well of a 16-well nucleofection cassette and nucleofected using the using E0-100 program with the 4D-Nucleofector Lonza Amaxa. After nucleofection, cells were transferred to 6 ml fresh cPIMDM and incubated at 37°C in 5% CO2 in air.